# Structural basis for control of integrative and conjugative element excision and transfer by the oligomeric winged helix–turn–helix protein RdfS

## TL;DR

This study reveals how the RdfS protein controls the excision and transfer of a bacterial DNA element involved in nitrogen-fixing symbiosis.

## Contribution

The study identifies a novel α1-helix in RdfS that enables oligomerization and indirect DNA recognition.

## Key findings

- RdfS binds to DNA regions in the IntS attachment site and its own promoter to regulate ICE excision.
- The α1-helix in RdfS is essential for oligomerization and DNA complex compaction.
- Removing the α1-helix abolishes RdfS-mediated ICE excision and conjugative transfer.

## Abstract

Winged helix–turn–helix (wHTH) proteins are diverse DNA-binding proteins that often oligomerize on DNA and participate in DNA recombination and transcriptional regulation. wHTH recombination directionality factors (RDFs) associated with tyrosine recombinases, stimulate excision of prophage and integrative and conjugative elements (ICEs). RdfS is required for excision and conjugation of the Mesorhizobium japonicum R7A ICE, ICEMlSymR7A, which carries genes for nitrogen-fixing symbiosis. We show RdfS binds to DNA regions within the IntS attachment site (attP) and within the rdfS promoter, enabling RdfS to coordinate rdfS/intS expression and stimulate RdfS/IntS-mediated ICEMlSymR7A excision. Several RdfS DNA-binding sites were identified. However, no consensus motif was apparent and no individual nucleotide substitutions in attP prevented RdfS binding. RdfS forms extensive helical filaments in crystals, with subunits contacting via a novel α1-helix absent in other wHTH-RDFs. RdfS oligomerized in solution in the absence of DNA. Molecular dynamics simulations supported a role for the α1-helix in oligomerization and compaction of nucleoprotein complexes. Removal of RdfS-α1 did not eliminate DNA-binding in vitro but reduced oligomerization and abolished RdfS-mediated ICEMlSymR7A excision and conjugative transfer. We propose the novel RdfS-α1 mediated oligomerization enables RdfS to specifically recognize larger DNA regions with low primary sequence conservation through an indirect readout mechanism.

Graphical Abstract